Sonny's Research ToDo:
safety training (see Required Safety Training link to the left)
(print out TxClass training history sheet and hole punch and plate into VDS sign in binder with others)
Get a lab notebook
make 250 ml of LB
make 4 LB/Agar +AMP plates
Nanodrop plasmid
Submit pGBR22 plasmid to DNA sequencing
Transformation efficiency
Analyze DNA sequence exercise (on computer)
RE digest and Agarose gel check
1st PCR - of pGBR22
2nd PCR - Red or Green PCR
3rd PCR - pLIC
Start Cloning of gene for Target
PASTE YOUR RESULTS IMAGES WITH CAPTIONS HERE (and include all in lab notebook):
Transformation Efficiency
Sonny Cabrera (sc38763) Wednesday, June 29, 2011 25ng pGBr22/ 7colonies
pGBR22 5ng/ 11 colonies
pGBR22 1ng/ 6 colonies
Submitting DNA to DNA Sequencing Facility- Results
Sonny Cabrera (sc38763) Wednesday, 28, 2011
pGBR22 Forward Sequence
Submitted Analysis to Google Docs
PCR Protocol: Trial 1 SP6/T7
Sonny Cabrera Friday July 1, 2011. Lane1:Skip Lane2: 5ul Marker Lane3:SampleA Lane4:SampleB Lane 5:SampleC Lane 6:SampleD Lane 7: Mystery Liquid/Possible Blue Juice. Ran on well for 40min on 105V. After results displayed a small neon blue spot near the samples.
Sonny Cabrera Friday July 8, 2011 Lane1:Skip Lane2: 5ul 100bp Marker Lane3:SampleA Lane4:SampleB Lane5:SampleC Lane6:SampleD Failure possibly due to pipeing techniqe. Retrial number 4 on Monday July 11, 2011.
PRC Protocol: Trial 4 SP6/T7 (Success)
Sonny Cabrera Wednesday July 13, 2011 Lane1:Skip Lane2: 5ul 100bp Marker Lane3:SampleA Lane4:SampleB Lane5:SampleC Lane6:SampleD Sucess acheived through a more careful pipetting technique.
Protocol of PCR for RE Cloning Trial 1
Sonny Cabrera Wednesday July 13 Lane1: Skip Lane2:100bp marker Lane 3:Sample1 Lane4:Sample2 Lane 5 Sample3 Lane6:Sample4 Lane7:Sample5 Lane8:Sample6 Lane9:Sample7 Lane10:Sample8 Results seem to
Protocol of PCR for RE Cloning Trial 2
Sonny Cabrera Thursday July 14 Lane1: Skip Lane2:100bp marker Lane 3:Sample1 Lane4:Sample2 Lane 5 Sample3 Lane6:Sample4 Lane7:Sample5 Lane8:Sample6 Lane9:Sample7 Lane10:Sample8
Protocol of PCR for RE Cloning Trial 3
Protocol of PCR for RE Cloning Trial 4
Protocol of PCR for RE Cloning Trial 5
Protocol of PCR for RE Cloning Trial 6 (Possible sucess)
Lane1: skip Lane2:!00 bp Marker Lane3:Sample1 Lane4:Sample2 Lane5:Sample3 Lane6:Sample4 Lane7:Sample 5 Lane 8:Sample6 Lane9: Sample 7 Lane10: Sample 8 Lane 11: Sample 9 Possible reasons for not being able to see the M13 for/rev set is due to spinning the samples prior to adding Taq and due to other factors such as PCR machine heat. Other research pages contain the protocol for PCR RE Red and not green. It could be possible that RE Red (pmCherry) takes a while longer to clone or is not vsible in comp with 100bp marker.
PRC Protocol for pLIC Seqencing vectors of pNIC-Bsa4
Trial 1 Results for Trials 1&2 of the PCR Protocol for PLIC Sequencing Vectors pNIC-Bsa4
could be as a result of poor mixing in the dilutions.
PRC Protocol for pLIC Seqencing vectors of pNIC-Bsa4 Trial 1
PRC Protocol for pLIC Seqencing vectors of pNIC-Bsa4 Trial 2 Success
Sonny Cabrera Monday August 1, 2011 Lane1:Skip Lane2:100bp (Should have been 1k bp) Lane3:SampleA Lane4:SampleB Lane5:SampleC Lane6:SampleD Lane8&9:Ben's pNic-BSA4 Cloning
Sonny - what are each of these lanes? - Dr. B
Protocol of PCR for pNIC-Bsa4 Cloning Trial 1 (Success)
Sonny Cabrera Monday August 3, 2011 Lane1:Skip Lane2:100bp Lane3:SampleA Lane4:SampleB Lane5:SampleC Lane6:SampleD Lane7:SampleE Lane8:SampleF Lanes with samples C D and E appeared to be brightest. Optioin B choosen from protocol.
Trial 2
Sonny Cabrera Monday August 3, 2011 Lane1:Skip Lane2:100bp Lane3:Sample1 Lane4:Sample2 Lane5:Sample3 Lane6:Sample4 Lane7:Sample5 Samples 2,4,and5 appeared brightest. Redo of protocol:Not enough Sample in PCR tubes.
PCR's at regular protocol requirements work well. Past three attempts at regular
protocol turn out well. All attempts at multiplying the samples to 50ul have
resulted in a unsuccessful result.
ToDo:
safety training (see Required Safety Training link to the left)
(print out TxClass training history sheet and hole punch and plate into VDS sign in binder with others)
Get a lab notebook
make 250 ml of LB
make 4 LB/Agar +AMP plates
Nanodrop plasmid
Submit pGBR22 plasmid to DNA sequencing
Transformation efficiency
Analyze DNA sequence exercise (on computer)
RE digest and Agarose gel check
1st PCR - of pGBR22
2nd PCR - Red or Green PCR
3rd PCR - pLIC
Start Cloning of gene for Target
PASTE YOUR RESULTS IMAGES WITH CAPTIONS HERE (and include all in lab notebook):
Transformation Efficiency
Submitting DNA to DNA Sequencing Facility- Results
Sonny Cabrera (sc38763) Wednesday, 28, 2011
pGBR22 Forward Sequence
Submitted Analysis to Google Docs
PCR Protocol: Trial 1 SP6/T7
PCR Protocol :Trial 2 SP6/T7
PCR Protocol: Trial 3 M13 For/Rev
PRC Protocol: Trial 4 SP6/T7 (Success)
Protocol of PCR for RE Cloning Trial 1
Protocol of PCR for RE Cloning Trial 2
Protocol of PCR for RE Cloning Trial 3
Protocol of PCR for RE Cloning Trial 4
Protocol of PCR for RE Cloning Trial 5
Protocol of PCR for RE Cloning Trial 6 (Possible sucess)
PRC Protocol for pLIC Seqencing vectors of pNIC-Bsa4
Trial 1 Results for Trials 1&2 of the PCR Protocol for PLIC Sequencing Vectors pNIC-Bsa4
could be as a result of poor mixing in the dilutions.
PRC Protocol for pLIC Seqencing vectors of pNIC-Bsa4 Trial 1
PRC Protocol for pLIC Seqencing vectors of pNIC-Bsa4 Trial 2 Success
Sonny - what are each of these lanes? - Dr. B
Protocol of PCR for pNIC-Bsa4 Cloning Trial 1 (Success)
Trial 2
Trial 3
PCR's at regular protocol requirements work well. Past three attempts at regular
protocol turn out well. All attempts at multiplying the samples to 50ul have
resulted in a unsuccessful result.
FALL FRI 2011
PCR Primer Design for Overlap Assembly - 10.14.11